Two-photon fluorescence imaging and specifically biosensing of norepinephrine on a 100-ms timescale

Norepinephrine (NE) is a key neurotransmitter in the central nervous system of organisms; however, specifically tracking the transient NE dynamics with high spatiotemporal resolution in living systems remains a great challenge. Herein, we develop a small molecular fluorescent probe that can precisely anchor on neuronal cytomembranes and specifically respond to NE on a 100-ms timescale. A unique dual acceleration mechanism of molecular-folding and water-bridging is disclosed, which boosts the reaction kinetics by ˃105 and ˃103 times, respectively. Benefiting from its excellent spatiotemporal resolution, the probe is applied to monitor NE dynamics at the single-neuron level, thereby, successfully snapshotting the fast fluctuation of NE levels at neuronal cytomembranes within 2 s. Moreover, two-photon fluorescence imaging of acute brain tissue slices reveals a close correlation between downregulated NE levels and Alzheimer’s disease pathology as well as antioxidant therapy.


Responses to the reviewers' comments
Reviewer #1: The work by Mao et al. reported a new fluorescence probe to measure norepinephrine. Measuring NE is a significant area of research. The proposed new dyes seem promising. The following concerns need to be addressed.
Comment 1: Fluorescence spectra corresponding to Fig. 1c on selectivity should be provided, with similar format as Fig. 1a.
Response: We appreciate the reviewer's high evaluation, positive recommendation, as well as the valuable comments.
As suggested by the reviewer, we have now supplemented the original fluorescence response plots with regard to the selective experiments. As shown in Figure R1a, except for NE, the addition of other related neurotransmitters hardly affected the fluorescence emission of the probe. Then, each fluorescence variation rate caused by different analytes were calculated ((F0-Fi)/F0, where F0 referred to the original fluorescence intensity of the probe, and Fi referred to the fluorescence intensity of the probe upon addition of each analyte), and normalized to the value of NE response (referred to 100%).
The resulting relative selectivity diagram was shown in Figure R1b (same as Fig. 1c in the main text), which verified the superb selectivity of probe BPS3 toward NE.  Response: Thanks for the valuable suggestion. We have found a more comprehensive and precise literature and revised the statement about neuronal signaling between synapses. According to Ref. R1 (Nature 2014, 515, 293-297.) and the schematic diagram below ( Figure R2, from Ref. R1), although there are four hypotheses to describe the vesicle fusion and the subsequent neurotransmitter release process in the presynaptic neuron, namely, (i) Kiss and run, (ii) Full-collapse fusion, (iii) Ultra-fast endocytosis, and (iv) Bulk endocytosis, the general process of chemical signaling between neurons is widely recognized, i.e., when an electrical pulse reaches the tip of the neuronal axon, the vesicles there respond by moving to the synaptic membrane, merging with it and releasing the neurotransmitters, which then migrate to the neighboring neuron across the synaptic cleft, where they are bound to the receptors located in the postsynaptic neuronal membrane and initiate neuronal responses. Therefore, we have revised the statement as: " Signaling between neurons occurs when neurotransmitters are released from the presnaptic membrane, diffuse across the synapse to neighboring neurons, and bind to receptors on the postsynaptic neuronal membrane." This new statement has been highlighted in Line 3 to 6 on Page 3 in the revised manuscript, and Ref.  Comment 3: One critical parameter is limit of detection, the authors used "population standard deviation" to calculate, it is not clear how did the author calculate the population standard deviation.
In statistics, there is a way to estimate population standard deviation from the sample standard deviation.
Response: Thanks very much for the comment. We actually used the standard deviation (σ), which was written as the population deviation by mistake. In general, the limit of detection (LOD) was calculated as follows: LOD = 3σ / S, where σ is the standard deviation of the measured fluorescence intensities of the probe solution at 480 nm: where, n = 20, is the fluorescence intensities of the probe of each measurement, ̅ is the averaged value for , thus σ is calculated to be 0.00055.
S is the slope of calibration curve (0.0033), so the LOD was calculated: We have corrected the statement and highlighted in Line 2-3 from the bottom on Page 6, in the revised manuscript. The calculation details were added and highlighted in the second paragraph on Page 3 in the revised Supplementary Information. The fast fluorescence response of the probes toward NE was carried out by using a stopped-flow accessory with a pneumatic drive system ( Figure R3a) (SFA-20, HI-Tech, TgK Scientific, United Kingdom), combined with a fluorescence spectrometer (Hitachi F-4600, Japan). As schemed in Figure   R3b, equal volumes of the probe solution (10 μM, 150 μL, syringe A) and pure water (150 μL, syringe B) were rapidly driven from syringes into a highly efficient mixer and the basal fluorescence intensity of the probe was monitored at 480 nm. Subsequently, NE (200 nM, 150 μL, syringe B') and the probe (10 μM, 150 μL, syringe A) solutions were rapidly driven from both syringes to the mixer in the same way to initiate the fast reaction. The resultant reaction volume then displaced the contents of the optical cell (5 μM, 300 μL of the probe solution), thus filling it with freshly mixed reagents. During the entire mixing process, the fluorescence intensity was continuously monitored. All the injected volume was limited by a stop syringe that provided the "stopped-flow". The dead time of such mixing system was ca. 8 ms, and the fluorometer has a shorter sampling interval (5 ms). So, in principle, any dynamic processes longer than this timescale can be monitored by this instrument, ensuring sufficient temporal resolution to measure the fluorescent response of the developed probes toward NE.
We have added and highlighted these experimental procedures to Section 1.2, on Page 2 in the revised Supplementary Information, and the instrument was also added and highlighted in Lines 6-8, on Page 23 in the revised manuscript. We have added highlighted this discussion in Lines 7-10, on Page 11, in the revised manuscript, and added Figure R4 as Figure S47 on Page 27 in the revised Supplementary Information. Comment 5: Page 16: the authors stated that "excellent ability in neuronal cytomembrane targeting, which may be derived from the strong affinity between the positively charged pyridiniums in BPS3 and the negatively charged cell membrane". It is not clear which part of the cell membrane is BPS3 attached to? How would this affect the spatial resolution in NE study?
Response: Although our confocal microscope could not provide higher resolution to see where exactly the probe was localized in the cell membrane, a high Pearson's coefficient value of 0.93 was obtained through the co-localization experiment with the commercial cell membrane tracer DiI ( Figure   R5a), indicating that they had very similar targeting capabilities. Given their similar amphiphilic structures, with positively charged heads and lipophilic tails ( Figure R5b), it is understandable that they have similar targeting capabilities that can uniformly label the entire neuron membrane. R2 On the other hand, by overlaying the fluorescent and bright field images of neurons, we could also observe that the dye was evenly distributed on the membrane of neurons. Other probes with similar pyridinium structures have also been reported to have cell membrane labeling ability. R3,R4 It is proposed that the cationic pyridinium have electrostatic affinity with the phosphate anion of the cell membrane surface, while the hydrophobic phenyl groups can be embedded into the cell membrane ( Figure R5c). Therefore, the current experiments have clearly demonstrated that the probe possesses general membrane labeling ability, while it is not yet possible to tell whether it has a more precise submembrane spatial resolution.
We have added highlighted this discussion in Lines 3-6 from the bottom, on Page 18 in the revised manuscript, and added Figure   Response: In order to understand the source of the fluorescence and its variation, we further synthesized two reference compounds, namely R1, R2 ( Figure R6a), which represented the two fragment moieties of the probe BPS3, respectively. As shown in Figure R6b, the p-bromophenyl pyridinium moiety (R1) of the probe emitted little fluorescence, while the S-phenyl carbonothioatecontaining pyridinium moiety (R2) exhibited similar fluorescence emission to the probe BPS3.
Therefore, it could be inferred that the fluorescence of the probe was generated from the S-phenyl carbonothioate-containing pyridinium moiety. Next, in order to rationalize the fluorescence response caused by the cleavage reaction triggered by NE, we further synthesized the third reference compound (R3) representing the fragment moiety of the product BPS3-OH ( Figure R6a). It could be seen from We have added and highlighted this discussion in the second paragraph on Page 9 and Page 10 in the revised manuscript. We also added the synthesis procedures and characterizations of the newly synthesized reference compounds R2, R3 on Pages 19-21, and added Figure R6 as Figure S44, and Figure R7 as Figure    Response: Thanks for the suggestion, we have conducted a more detailed discussion on the imaging of brain tissue slices, as follows: We prepared acute brain tissue slices from four different regions of both AD and normal mice, namely, cornu ammonis of hippocampus (CA1), primary somatosensory cortex (S1BF), laterodorsal thalamic nucleus (LD), and caudate putamen (CPu), and then cultured with the probe BPS3. Confocal microscopic images of these tissue slices were presented in Figure R8a (same as Fig 4f in the   manuscript), where the pseudocolor ranging from blue to red represented increase of the relative fluorescence intensity. Then, by randomly selecting 25 cells in each image, followed by statistical analysis on their fluorescence intensity variations, the histogram was generated as Figure R8b. The statistical results indicated some inhomogeneity of NE distribution in various brain regions. It is worth noting that under the AD model, the NE content in all these brain regions decreased significantly to 58.3%, 48.0%, 58.0%, and 63.9% of the normal level, in CA1, S1BF, LD, and Cpu regions, respectively. This downregulated NE level might be due to the reduced noradrenergic activity caused by the oxidative damage of neurons in the brain of AD mouse. Thereafter, we further incubated the AD mouse brain slices with an antioxidant drug N-acetylcysteine (NAC). As shown in Figures R8a, after NAC treatment, the fluorescence intensities of all the four regions were decreased as compared to AD samples, indicating the elevated concentrations of NE in the AD brain slices after NAC treatment, which returned to 87.2−96.8% of normal levels, and among these four regions, the S1BF region had the most significant increase in ratio ( Figure R8b). These results suggested a potential effect of the antioxidant NAC in improving the noradrenergic activity of neurons, which might be beneficial in relieving AD pathology.
We have added and highlighted this discussion in Lines 3-17, on Page 21, in the revised manuscript. Figure R8. (a) Two-photon fluorescence images of the BPS3-incubated tissue slices from CA1, S1BF, LD, and CPu regions of normal and AD mouse brains, as well as the NAC-treated AD mouse brain.
Comment 8: Typo, abstract, "response" should be "respond" Response: Thanks for the correction. We have corrected this typo and highlighted in Line 5 on Page 2 in the revised manuscript.
Comment 9: Experimental procedure on measuring reaction kinetics should be provided. "It was boosted by >10 5 times by the new probe", why?
Response: For the detailed experimental procedures of the kinetics studies, please see the response to Comment 4. Regarding the acceleration rate (>10 5 ) of the reaction rate induced by molecular conformational folding, we compared the reaction kinetics of the probe BPS3 and the control compound BPS2, respectively. As confirmed in the main text, probe BPS3 possessed a folded conformation, while for the control compound BPS2, there were only two carbons in the alkyl chain between the two rigid phenyl groups, thus it could hardly form a folded conformation due to the larger molecular bond tension and steric hindrance ( Figure R9a). Whereafter, the reaction kinetics of these two compounds with NE were subsequently studied. It was found that despite with only one-carbon shorter in the alkyl chain, compound BPS2 hardly produced any response to NE at room temperature, even extending the reaction time to 7 days ( Figure R9b). Even if the temperature was elevated to 50 o C, it still could not achieve the reaction equilibrium within 12 hours ( Figure R9c). In order to get a better kinetic fitting curve, we continued to increase the temperature to 60 o C, which could reach the reaction equilibrium within 8 hours ( Figure R9d), so that we were able to calculate a kobs value of 0.5818 h -1 (equal to 1.6×10 -4 s -1 ) for the reaction between BPS2 and NE at 60 o C ( Figure R9e). In contrast, the probe BPS3 that possessed a highly similar structure but with folded conformation, could complete the reaction to NE within 100 ms ( Figure R9f), thus the kobs value was calculated to be 0.0532 ms -1 (equal to 53.2 s -1 ) ( Figure R9g). Therefore, by comparing the kobs values for the two probes with NE, the acceleration rate could be calculated as: 53.2 / (1.6×10 -4 ) = 3.325×10 5 . Moreover, this acceleration rate was calculated by using the kobs values of the reaction for BPS2 at 60 o C (because no reaction was detected at room temperature, Figure R9b), while for BPS3 at room temperature.
Therefore, the actual acceleration rate at room temperature would be much higher than 10 5 times. is not non-invasive. The statement the authors mentioned in the introduction about "non-invasively monitor biomolecules" is misleading.
Response: Yes, thanks for the comment and we agree with the reviewer that fluorescence imaging technology itself can be non-invasive, and this statement only makes sense for in vivo experiments.
However, most currently reported fluorescent probes (including the probe in this paper) are applied at the level of cells or tissue slices.
Therefore, we have removed the statement of "non-invasive" and revised the statement as follows: "Fluorescent probes combined with fluorescence imaging technology may serve as a promising candidate to monitor biomolecules, including neurotransmitters, in real-time with high sensitivity and spatiotemporal resolution." It was highlighted in Lines 7-9 from the bottom, on Page 3, in the revised manuscript.
Reviewer #2: In this work, Tian and co-workers developed a simple yet efficient small molecular fluorescent probe that could precisely anchor to the neuronal membrane and specifically respond to NE on a 100-mstimescale. More importantly, they proposed a unique dual-acceleration mechanism, and disclosed the important role of the molecular-folding and the transition state of a water-bridged six-membered ring, which is a quite interesting finding. This is the first paper to systematically study the kinetics of NE detection, and provides a small-molecular tool to capture transient NE fluctuations, which will facilitate the research of complex neurotransmission with high spatiotemporal resolution. Therefore, I recommend the urgent publication in Nature Communications.
Comment 1: Considering that the probe molecule consists of two non-conjugated aromatic moieties, so which group does the fluorescence emission come from?
Response: We very much appreciate the reviewer's high evaluation and positive recommendation on our manuscript.
To answer this question, we further synthesized two reference compounds, namely R1, R2 ( Figure   R10a), which represented the two fragment moieties of the probe BPS3, respectively. As shown in Figure R10b, the p-bromophenyl pyridinium moiety (R1) of the probe emitted little fluorescence, while the S-phenyl carbonothioate-containing pyridinium moiety (R2) exhibited similar fluorescence emission to the probe BPS3. Therefore, it could be inferred that the fluorescence of the probe was mainly generated from the S-phenyl carbonothioate-containing pyridinium moiety of the probe.
We have added and highlighted this discussion in the second paragraph on Page 9 in the revised manuscript. We also added the synthesis procedures and characterizations of the newly synthesized reference compounds R2, R3 on Pages 19-21, and added Figure R10 as Figure   Comment 2: In order to confirm the influence of molecular folding on reaction kinetics, the authors designed and synthesized a series of control compounds. Here I suggest whether it is possible to prepare a "half-molecule" probe containing the reaction site, to see how the reaction rate is. I think this may further support the author's inference.
Response: Thanks very much for the suggestion. We have synthesized the control compound R2 as the "half-probe" and studied its reaction kinetics toward NE and compared it with the probe BPS3. As shown in Figure R11a, although it had the same reaction site as BPS3, the reaction rate of the control compound R2 toward NE was much lower than that of the probe BPS3. Under the same conditions, it took about 257 seconds to reach the reaction equilibrium, while only 100 ms for the probe BPS3 ( Figure R11b), a difference of ca. 2570 times. Therefore, this experiment again verified the important role of the unique folded conformation of BPS3 in accelerating the reaction kinetics.
We have added and highlighted this discussion in Lines 10-15, on Page 14, in the revised manuscript.
We have also added Figure R11a as Figure S50 on Page 29, in the revised Supplementary Information. Reviewer #3: The authors developed a novel probe (BPS3), which selectively reacted with norepinephrine (NE) on 100-ms timescale. The mechanism of the fast and selective reaction was carefully investigated by spectroscopic measurements and density functional theory calculation. The authors also demonstrated the applicability of the probe to monitoring NE release in living neurons and mouse brains. The data and results are interesting. However, some deficiencies in experimental explanations exist in the current manuscript.
Comment 1: In Fig. 4b, the authors evaluated the response of BPS3 by monitoring DiI signals. The authors should explain why they did not monitor BPS3 directly.
Response: We very much appreciate the reviewer's high evaluation and positive recommendation on our manuscript, as well as the valuable comments that greatly helped us improve this manuscript.
Regarding the fluorescence signal in Figure 4b, in fact it was the fluorescence signal from the probe BPS3, but we used a red pseudocolor, which might mislead the readers as it looked like the DiI signal in Figure 4a. Therefore, as shown in Figure R12 below, we have modified the imaging color of the neurons to green color for ease of reading.
This modified Figure 4b was highlighted on Page 19, in the revised manuscript. intensities were significantly attenuated in the first 2 seconds (0−2 s) compared with that without high potassium stimulation (PBS only). Thereafter, the fluorescence signals hardly changed any more (2−10 s). It indicated that neurons could respond to external stimuli of high potassium and reach a steady state in a quite short time (within 2 s). However, the total fluorescence variation rate (∆F/F0) was only ~40%, and the concentration of norepinephrine was estimated to be ~100 nM from the in vitro working curve (Fig. R2c). Therefore, the overall fluorescence intensity changes might not be very obvious from the naked eye, but still significant enough to be read by the microscopic instrument (Fig. R2b).